A KRAS-variant in ovarian cancer acts as a genetic marker of cancer risk

Page 1

Mole

A KMar

ElenaRacheHarveDanie

Abst

Intro

Ovalogic mgynecoitheliacompofallopiof canthan 1

AuthorObstetHealthCounseMediciBiologyObstet8DepaPhilade

Note: SResear

Correspeutic RNew Hayale.ed

doi: 10

©2010

www.a

Canceresearch

cular and Cellular Pathobiology

RAS-Variant in Ovarian Cancer Acts as a Genetic

R

ker of Cancer Risk

Ratner1, Lingeng Lu2, Marta Boeke3, Rachel Barnett4, Sunitha Nallur3, Lena J. Chin6, Cory Pelletier3,l Blitzblau3, Renata Tassi7, Trupti Paranjape3, Pei Hui5, Andrew K. Godwin8, Herbert Yu2,

y Risch2, Thomas Rutherford1, Peter Schwartz1, Alessandro Santin1, Ellen Matloff4, l Zelterman2, Frank J. Slack6, and Joanne B. Weidhaas3

ractOva

diseasand altumoriant althis opatienheredithat thmarkeyses. LBRCA2

cer death5,000 wom

s' Affiliationrics and Gy, 3Departmling, and 5

ne; 6Depar, Yale Unirics and Gyrtment oflphia, Penn

upplementch Online (

ponding Aadiology, Yven, CT 06u; Frank J. S

.1158/0008-

American A

acrjourna

rian cancer (OC) is the single most deadly form of women's cancer, typically presenting as an advancede at diagnosis in part due to a lack of known risk factors or genetic markers of risk. The KRAS oncogenetered levels of the microRNA (miRNA) let-7 are associated with an increased risk of developing solids. In this study, we investigated a hypothesized association between an increased risk of OC and a var-lele of KRAS at rs61764370, referred to as the KRAS-variant, which disrupts a let-7 miRNA binding site inncogene. Specimens obtained were tested for the presence of the KRAS-variant from nonselected OCts in three independent cohorts, two independent ovarian case-control studies, and OC patients withtary breast and ovarian cancer syndrome (HBOC) as well as their family members. Our results indicatee KRAS-variant is associated with more than 25% of nonselected OC cases. Further, we found that it is ar for a significant increased risk of developing OC, as confirmed by two independent case-control anal-astly, we determined that the KRAS-variant was present in 61% of HBOC patients without BRCA1 ormutations, previously considered uninformative, as well as in their family members with cancer. Our

gs strongly support the hypothesis that the KRAS-variant is a genetic marker for increased risk of de-

findinveloping OC, and they suggest that the KRAS-variant may be a new genetic marker of cancer risk for HBOCfamilies without other known genetic abnormalities. Cancer Res; 70(16); 6509–15. ©2010 AACR.

Thecept fowith hited rirelativthan tfamiliwith t

duction

rian cancer (OC) is the second most common gyneco-alignancy, but the most common cause of death fromlogic malignancies (1). More than 90% of OCs are ep-l OCs, which are believed to arise from the epithelialnent of the ovary or from the fimbriated end of thean tubes (2). Epithelial OC is the fifth leading cause

in females in the United States, with moreen dying yearly of this disease (1).

Onefamilidrometumordiscovadvangenes(5). Hmutatformaicantnot beremaitionssuscepAlth

genesways aOC (7)

s: 1Division of Gynecologic Oncology, Department ofnecology, 2Department of Epidemiology and Publicent of Therapeutic Radiology, 4Cancer GeneticDepartment of Pathology, Yale University School oftment of Molecular, Cellular, and Developmentalversity, New Haven, Connecticut; 7Department ofnecology, University of Brescia, Brescia, Italy; andMedical Oncology, Fox Chase Cancer Center,sylvania

ary data for this article are available at Cancerhttp://cancerres.aacrjournals.org/).

uthors: Joanne B. Weidhaas, Department of Thera-ale University School of Medicine, 333 Cedar Street,520. Phone: 203-737-2165; E-mail: joanne.weidhaas@lack, E-mail: [email protected].

5472.CAN-10-0689

ssociation for Cancer Research.

ls.org

re are few identified risk factors for epithelial OC, ex-r aging. More than 90% of cases arise after menopause,alf of all OC cases diagnosed after age 63 (3). An inher-sk component exists for developing OC, as first-degreees of OC patients more frequently develop the diseasehe general population, whose risk is 1/71 (4). Knownal cases make up approximately 10% of all OC cases,he rest considered sporadic.of the best-studied hereditary syndromes linked to

al OC is hereditary breast and ovarian cancer syn-(HBOC). HBOC is associated with mutations in thesuppressor genes BRCA1 and BRCA2. Although theery of BRCA1 and BRCA2 represented a significantce in understanding familial OC predisposition, theseaccount for less than half of familial excess risk of OCBOC patients who test negative for BRCA1 and BRCA2ions are referred to as “mutation negative” or “unin-tive.” Additional gene alterations explaining any signif-proportion of the remaining familial risk for OC haveen identified, and it has been hypothesized that thening genetic risks for familial OC are due to combina-among many loci of alleles that confer low-penetrancetibility (6).ough common cellular tumor suppressors and onco-such as p53, AKT, RAS, c-MYC, and their associated path-

re thought to cooperate to lead to the development of, there have been no known germline mutations in these

6509

Page 2

genessinglemoderBRAF,of OChistoloderlingenomthousathe geallelesallelesThe

risk inreflectied arcommSNPsThe tabeen fcountthe stcandidto findtive poMic

codingby negtially(3′UTRin virtoncogglobalvels ochangshowscan berisk wacterisThe

portanOC only idengene (the KRin Eascans, alationmoretions (shownto incvarianincreain twoto 20%U.S. etpeople

normthe Kwhichoutcoto becancerBec

humaKRAS-KRAS-with otionsIn conof patwe hemarkeulatioated wof thomembthe KRand mfamili

Mate

SampSam

tal we(n = 1fixed t2007BecauKRAS-normaline Ddata wof cantion, wOC

the YadividustatistCon

Hospiates oAll cothe cononmand fa

SampBet

patients who underwent surgery for ovarian tumors at theDepartment of Gynecology, Gynecologic Oncology Unit, at

Ratner et al.

Cance6510

previously associated with OC. A study using a tagging-nucleotide polymorphism (SNP) approach to identifyate/low-risk susceptibility alleles of the proto-oncogenesERBB2, KRAS, NMI, and PIK3CA (8) found no evidenceassociation with these SNPs. Only when stratified bygic subtype did one common variant allele have bor-e evidence of association with epithelial OC (8). A recente-wide association study (GWAS) of tagging SNPs innds of OC patients and controls identified a region ofnome associated with OC; however, the studied variantin this region were not within known genes, and thesepredicted a decrease in OC risk (9).lack of the identification of powerful biomarkers of OCthese studies using the tagging SNP approach likelys inherent limitations of this approach; the SNPs stud-e not representative of all SNPs, do not capture lesson variants, and are rarely functional, but rather arethat act as markers identifying common haplotypes.gging SNPs associated with disease risk thus far haveound to have only modest predictive power and ac-for a small fraction of heritability. Many believe thatudy of functional and less heterozygous SNPs throughate gene studies or more focused analyses is necessaryimportant, less common variants with greater predic-wer (10–12).roRNAs (miRNA) are a class of ∼22-nucleotide non-RNAs that are evolutionarily conserved and functionatively regulating gene expression by binding to par-complementary sites in the 3′ untranslated regions) of target mRNAs. MiRNAs are aberrantly expressedually all cancers, where they function as a novel class ofenes or tumor suppressors (13). Because miRNAs aregene regulators, even small aberrations in miRNA le-r their target sites can lead to important cellulares. In support of this concept, emerging evidencethat SNPs within miRNAs or miRNA binding sitesfunctional and act as powerful biomarkers of cancer

hen one allele alters miRNA function or binding char-tics (14–16).let-7 family of miRNAs has been shown to play an im-t role in many cancers (17–19). let-7 regulates multiplecogenes, including KRAS and c-MYC (20). We previous-tified a germline SNP in the 3′UTR of the KRAS onco-rs61764370). The variant, derived allele, referred to asAS-variant, is relatively uncommon: It is almost absentt Asians and Native Americans, is uncommon in Afri-nd has a minor allele frequency of about 7% in popu-s of European descent, based on the genotyping ofthan 2,400 samples representing 46 geographic popula-14). The KRAS-variant is functional and was previouslyby us to disrupt binding of let-7 to KRAS, leading

reased KRAS levels in in vitro assays (14). The KRAS-t was initially found to be a genetic marker ofsed susceptibility to non–small-cell lung cancer (NSCLC)U.S. case-control studies, where it was identified in 18%of NSCLC patients versus 12% to 14% of noncancerous

hnicity-matched control populations (14). Of note, inthe KRAS-variant is almost always heterozygous in both 9 F. Slac

r Res; 70(16) August 15, 2010

al and tumor tissues. In addition, lung tumors withRAS-variant were found to have lower let-7 levels,has previously been shown to be a biomarker of poorme (21). The KRAS-variant was subsequently showna genetic marker of poor outcome in head and neck(22).ause of the importance of both let-7 and KRAS inn solid tumors, we evaluated the frequency of thevariant in patients with other solid tumor types. Thevariant was present in fewer than 18% of all patientsther solid tumor types tested and in control popula-matched for ethnicity (n > 17,000 individuals tested).9

trast, the KRAS-variant is present in more than 25%ients with epithelial OC. Through case-control analysis,re show that the KRAS-variant seems to be a geneticr of risk for developing OC for the general female pop-n (P < 0.020). Also, the KRAS-variant is strongly associ-ith uninformative HBOC families, being present in 61%se tested (n = 31), and segregates with their familyers with cancer (P < 0.001). These findings suggest thatAS-variant represents a new genetic marker of OC riskay account for genetic risk in a number of HBOC

es previously considered uninformative.

rials and Methods

les from New Haven, Connecticutples from patients with OC at Yale/New Haven Hospi-re recruited and collected from fresh frozen tissue2), DNA isolated from paraffin-embedded formalin-issue (n = 23), blood (n = 71), or saliva (n = 51) betweenand 2009 (total n = 157; Supplementary Table S2).se we have previously extensively validated that thevariant is not somatic but germline (identical in patient'sl and tumor tissues; ref. 14), we collected primarily germ-NA for these studies from either blood or saliva. Patientere collected including age, ethnicity, and family historycer. OC subtype was established by pathologic classifica-ith only epithelial OC cases included in this study.patients from HBOC families were recruited throughle Cancer Center Department of Genetics, and one in-al was included from each family as the index case forical analysis.trols (all female) were recruited from Yale/New Havental beginning in 2008 from healthy friends and associ-f patients; none were genetically related to the patient.ntrol DNA samples were derived from saliva. None ofntrols had any prior diagnosis of cancer (other thanelanoma skin cancer). Information on age, ethnicity,mily history was recorded.

les from Turin, Italyween October 1991 and February 2000, there were 264

k and J. Weidhaas, unpublished data.

Cancer Research

Page 3

the UnInstituItaliannosedOC oflial ovcludedavailathese

SampTum

100 paOncol2001 aof Eurical retientsor Matherapdebul(PFS).

Case-The

compl100 amatchovariemovalThe

Connethat pen froOC benew pa reprarea aods. CCasesanalysin this

StatisFor

used tgroupthe diWeinbSurvivzardswith OAll sta9.1.2 (

DetecDNA

tissue,

ticut cing. Tprimeniquessequeprevioheteroof anyformworkKRAS-

CalcuThe

paringcarryiby a lthe dibasedrisk ototal K

Resu

The KWe

sentedthe KRthis vaquencpopultestedpendea UnivTurinin thisand agThe frhighestudieNew HTo

risk owe pecontaicantlyriersconfidings wThe Cand 32of devate an lysis (OR, 1.7; 95% CI, 1.11–2.63, P = 0.016; Table 1).These indings suggest that the KRAS-variant may be a

MiRNA/KRAS–Based Genetic Marker of Ovarian Cancer Risk

www.a

iversity of Turin in Italy, and tissue was collected aftertional Review Board (IRB) approval. All patients wereof European ancestry. Of these patients, 23 were diag-with metastatic cancer, 19 with benign tumors, 6 withnonepithelial origin, and 1 with endometriosis. Epithe-arian tumors from the remaining 215 patients were in-in this study. Additional details on these samples are

ble (17). DNA from these samples was supplied forstudies.

les from Brescia, Italyor samples for DNA extraction were collected fromtients with epithelial OC at the Division of Gynecologicogy at University of Brescia, Italy, between Septembernd December 2008 after IRB approval. All patients wereopean ancestry. Clinical data were collected from med-cords and were available for all patients. Fifty-nine pa-were followed from the date of first surgery until deathy 5, 2009. Patients who received neoadjuvant chemo-y were excluded from nonstatic parameters such asking, residual disease, and progression-free survivalDNA from these samples was supplied for these studies.

control analysisYale cases and controls were selected from those withete information from Yale/New Haven Hospital (n =nd 101, respectively). All were women and wereed for age and ethnicity. For the controls that had theirs removed for benign reasons, their age at ovarian re-was recorded as their age of testing for this study.Connecticut case-control study was approved by thecticut Department of Public Health and all 32 hospitalsarticipated. Potential cases were English-speaking wom-m Connecticut, diagnosed at 35 to 79 years of age withtween September 1, 1998 and February 28, 2003, withrimary invasive epithelial ovarian tumors. Controls wereesentative sample of the general population of the studynd identified by list-based random digit dialing meth-ases and controls were matched for age and ethnicity.and controls with prior cancer were excluded from theis. Further details are available (23). DNA samples usedstudy included 320 cases and 328 controls.

tical methodsnumerical variables (such as age), linear models wereo compare the differences between case and controls. χ2 and exact methods were performed to determinestribution of ethnicity in cases and controls. Hardy-erg testing was analyzed using the ALLELE procedure.al analyses were performed using Cox proportional ha-regression model. The association of the KRAS-variantC was determined using logistic regression modeling.tistical analyses were performed using SAS versionSAS Institute).

tion of the KRAS-variant

was collected using standard isolation methods fromblood, buccal cell samples, or saliva. Only the Connec- 10 J. We

acrjournals.org

ase-control underwent DNA amplification before test-he KRAS-variant was assayed using an allele-specificr and a PCR-based TaqMan assay using standard tech-. Validation of this assay through duplicate testing andncing was previously performed and reported (14). Asusly shown, the KRAS-variant is almost always in thezygous state in its carriers, with less than 3% to 5%population containing the variant in the homozygous(14). We thus combined the two genotypes in thistogether and referred to both as people “carrying” thevariant.

lation of positive predictive valuepositive predictive value (PPV) is calculated by com-the percentages of KRAS-variant–carrying and non-

ng patients with OC and without OC and multiplyingifetime risk of 1.4% of developing OC to determinefference in lifetime cancer risk. Control frequency ison the Yale controls. PPV is then the lifetime cancerf KRAS-variant–carrying patients (with OC) over theRAS-variant–carrying people.

lts

RAS-variant and ovarian cancer risktested women diagnosed with epithelial OC who pre-at Yale/New Haven Hospital for surgery (n = 157) forAS-variant and discovered that more than 27% carriedriant allele. Because this was a significantly higher fre-y than previously shown in any normal or cancerousation [18% (refs. 14, 22) and >9,000 additional people),10 we validated this finding in two additional, inde-nt cohorts of epithelial OC patients. The first was fromersity Hospital in Northern Italy at the University of(n = 215), and 26% of patients carried the KRAS-variantcohort. The second was from Brescia, Italy (n = 100),ain 25% of these OC patients carried the KRAS-variant.equency of the KRAS-variant was thus significantlyr in these OC cohorts than in any group previouslyd, including noncancerous controls collected at Yale/aven Hospital (Fig. 1).investigate if the KRAS-variant predicts an increasedf developing OC for nonselected female populations,rformed case-control analyses. The Yale case-controlned 100 cases and 101 controls and showed a signifi-increased risk of developing OC for KRAS-variant car-by multivariate analysis [odds ratio (OR), 2.46; 95%ence interval (95% CI), 1.14–5.29; P = 0.020]. These find-ere validated in a second independent case-control:onnecticut OC case-control consists of 320 patients8 controls and also showed a significant increased riskeloping OC for the KRAS-variant carriers by multivari-

af

idhaas, unpublished data.

Cancer Res; 70(16) August 15, 2010 6511

Page 4

genetinonse

OvariWe

differealencehigheswith mWe

cific cKRAS-not a

tumorpresenor PFry Tabtientsthe KRstudy.Bec

KRAScodonn = 10nonmovariamutatcanceKRAS-acquir

AssocAs t

for spmarketo bewith ahad eidegreebreastunderthese13 wemative

Tabrisk

KRA

NonVariP

KRA

NonVariP

*Ad†Thecate

Figurecompatested for the presence of the KRAS-variant and frequency of carriersdepicte

Ratner et al.

Cance6512

c marker of an increased risk of developing OC inlected women.

an cancer variables and the KRAS-variantevaluated the distribution of the KRAS-variant in thent subtypes of epithelial OC. We found that the prev-of the KRAS-variant varied between subtypes, beingt in nonmucinous cancers and rarely found in patientsucinous OCs (P < 0.05; Table 2).studied a range of variables to see if there were spe-haracteristics segregating OC patients carrying the

d with the number tested in parentheses.

variant versus those without. We found that there wassig

36 (22

leo

case-contr

S Uni Mult

OR ( OR (

vaan

icut case-co

S Uni Mult

OR ( OR (

gory.

r Res; 70(16) August 15, 2010

grade, residual tumor size, debulking, stage of OCtation, response to platinum-based chemotherapy,S (hazard ratio, 1.12; 95% CI, 0.71–1.76; Supplementa-le S1A–E). The trend toward worse PFS for OC pa-carrying the KRAS-variant may suggest an effect ofAS-variant on OC outcome and may warrant further

ause the KRAS-variant is located in the 3′UTR of theoncogene, we tested available tumor samples for KRASmutations (n = 6 non–KRAS-variant-carrying patients,KRAS-variant–carrying patients). Not surprisingly, as

ucinous OC rarely has activated KRAS, none of then tumors tested had the common KRAS-activatingions. These observations agree with prior findings inr samples by us (14) as well as others (24) that thevariant is not enriched in tumors with other tumor-ed KRAS mutations.

iation of the KRAS-variant with HBOChe KRAS-variant seemed to be associated with OC riskoradic OC, to further validate its role as a geneticr of OC, we next examined OC patients consideredat high risk for having a familial genetic abnormalityfamily history consistent with HBOC. These patientsther personal and/or family histories (first- or second-relatives) of at least one additional case of OC and/orcancer, all were of European ancestry, and all had

gone BRCA mutation analysis. Sixty-seven patients fitparameters: 23 were positive for BRCA1 mutations,re positive for BRCA2 mutations, and 31 were uninfor-(BRCA1 and BRCA2 mutation negative). Overall, 8 of

%) of BRCA mutation carriers also were carriers of the

nificant difference in patient age at first surgery, KRAS-variant: 7 of 23 (30%) of BRCA1-mutant carriers and 1

1. Case-control re

sults indicate that the KRAS-variant may be a genetic marker for an increasedf developing OC

Cas

Cas

Cont

Cont

Cancer Res

Yale

ol

variate

95% CI)

ivariate*

95% CI)

e

rol

riant (T/T)

.00 .00 1 1 74 (74.0) 88 (87.3)t (G/G and G/T)† 2.38 (1.16–5.09) 2.46 (1.14–5.29) 26 (26.0) 13 (12.7)

0.02 0.02

Connect

ntrol

variate

ivariate* e rol

95% CI)

95% CI)

riant (T/T)

00 .00 va 1. 1 225 (73.1) 272 (84.5)ant (G/G and G/T)† 2.01 (1.36–2.99) 1.7 (1.11–2.63) 83 (26.9) 50 (15.5)

0.0005 0.016

justed for age and race.G/G genotype is found in less than 5% of cases and controls and is thus combined with the G/T genotype for the variant

1. The KRAS-variant is found frequently in patients with OCred with controls. Patients from three separate cohorts were

earch

Page 5

of 13 (tion osent aKRAS-Of t

(61%)highertion (1controfamily6.78%valueHBOC

FamilWe

knownilies wtive ffamilicarrieFina

a BRCKRAS-cance

FigureA line tindicate

Table lence o KRAS- e

Subty n Non–K KRAS

CC 22MU 15EN 52SPUNMC

NOTAbbMC

MiRNA/KRAS–Based Genetic Marker of Ovarian Cancer Risk

www.a

8%) of BRCA2 mutant carriers. The differential associa-f the KRAS-variant with BRCA1 and BRCA2 may repre-biological modification of BRCA penetrance by thevariant, a hypothesis that requires additional study.he 31 uninformative HBOC patients with OC, 19 of 31carried the KRAS-variant, a frequency significantlythan documented rates for either the healthy popula-4) or other OC patients (P < 0.0001, compared withl). For a KRAS-variant–carrying uninformative HBOCmember, this results in a PPV for developing OC of(95% CI, 5.78–7.76). In contrast, the negative predictive

, mucinous.

for a negative KRAS-variant test in an uninformative are un

hrough the symbol indicates the person has died. “d.” means dead, with the aged. Star indicates tested for the KRAS-variant and a carrier. hx, history; ca, canc

acrjournals.org

ies segregating the KRAS-varianttested at least two additional family members withcancer status in two of the uninformative HBOC fam-hose proband carried the KRAS-variant and was nega-or BRCA1 and BRCA2 mutations. In each of thesees, at least two relatives diagnosed with cancer alsod the KRAS-variant (Fig. 2; Supplementary Fig. S1).lly, we compared the pedigrees of HBOC families withA1 mutation (n = 11), a BRCA2 mutation (n = 8), or thevariant (n = 13) and recorded the demographics andr types in their family members. We found that there

ique familial profiles for each of these groups, which

family member is 99.37% (95% CI, 99.22–99.53). differ by ethnicity, cancer type, and age of cancer onset, with

2. An HBOC family with the KRAS-variant. Circles are women, squares are men. A black area within the symbol indicates the person had cancer.

2. Preva

f the variant in OC patients by subtyp

indicated. “dx” meaer.

pes

RAS-variant patients, T/T (%) -variant patients, G/T and G/G (%)

14 (63.6)

8 (36.4) 10 (66.7) 5 (33.3) 37 (71.2) 15 (28.9)

127 (76.1)

40 (23.9) 16737 30 (81.1) 7 (18.9)22 20 (90.9) 2 (9.1)

E: Patients from the Yale and two Italian studies where subtype was documented.reviations: CC, clear cell; MU, malignant mixed Mullerian; EN, endometrioid; SP, serous papillary; UN, undifferentiated;

ns diagnosed with cancer, with the age

Cancer Res; 70(16) August 15, 2010 6513

Page 6

KRAS-to be nat thepatien

Discu

Ourin therisk omore61% oered uport tmarkegest thAlth

varianthe KRthe tavarianis funculatesmostin prioof <7%GWASThus,with pble torisk thThe

to themenoppreviosuch a

genesgests trepairteratiogrowtprevioincrea(14), aage (2with nresentlead tsignalOC. BmiRNalter Kto unprequirAlth

ber ofof themembthe KRof newmativwill hawho topingformaGen

difficuvery fcasestory r

11 K. Kid

ureBogrbleOClikwn)mo

g cailiesAS-iag

h OC (P = 0.006).

Ratner et al.

Cance6514

variant ovarian families being significantly more likelyon-Jewish, have lung cancer in the family, and be oldertime of their OC diagnosis than BRCA-mutant OCts (Fig. 3).

ssion

results reveal that the variant allele at a polymorphismKRAS 3′UTR, the KRAS-variant, is associated with thef developing epithelial OC (OR, 2.46), is identified inthan 25% of nonselected OC patients, and is found inf OC patients from HBOC families previously consid-ninformative for gene mutations. These findings sup-he hypothesis that the KRAS-variant is a new geneticr of an increased risk of developing OC and also sug-at this allele of KRAS may be a new HBOC locus.ough it may seem surprising that a single-nucleotidet could have such predictive power for disease risk,AS-variant represents an entirely different entity thangging SNPs studied and used in GWAS. The KRAS-t was identified through a candidate-gene search. Ittional and disrupts a let-7miRNA binding site that reg-the important human oncogene, KRAS (14). Perhapsimportantly, the KRAS-variant has not been includedr GWAS platforms and has a minor allele frequency, whereas surrounding KRAS SNPs studied in theplatforms have a minor allele frequency of ≥20%.11

even if the KRAS-variant is in linkage disequilibriumreviously studied SNPs in this region, it was not possi-determine the association of the KRAS-variant with OCrough these prior studies.OC in KRAS-variant carriers has a similar phenotypemajority of epithelial OC and occurs primarily in post-ausal women. This is unlike the OC associated with

sly identified inherited genetic markers of OC risk,BRCA mutations, which disrupt DNA repair pathway

harboassoccan bdiseas

us

reinfocanced, personal communication.

r Res; 70(16) August 15, 2010

and are associated with early-onset cancer. This sug-hat the KRAS-variant may not act through altered DNA, but perhaps instead creates an environment where al-ns that occur normally with aging allow aberrant cellh and oncogenesis. In support of this hypothesis, weusly reported that the KRAS-variant is associated withsed KRAS levels in the background of lower let-7 levelsnd others have shown that let-7 levels decrease with5). Although KRAS mutations have not been associatedonmucinous epithelial OC, the KRAS-variant may rep-a novel form of KRAS activation and overexpression oro disruption of the epidermal growth factor receptoring pathway, a pathway frequently misregulated inecause the KRAS-variant, like disruption of otherA binding sites in the 3′UTR of KRAS (20), does notRAS mRNA levels, and because we did not have accessrocessed tumor tissue for our studies, these hypothesese further validation in other patient cohorts.ough our study is somewhat limited by the small num-uninformative HBOC patients, the frequent associationKRAS-variant with these patients and their familyers with cancer further strengthens the hypothesis thatAS-variant is a genetic marker of OC risk. Identificationsuch markers of OC risk is critical for these uninfor-

e families, as those who test positive in these familiesve a confirmed increased inherited risk, whereas thoseest negative will in fact be at a decreased risk of devel-OC compared with the general female population, in-tion that will be equally valuable to them.etic risk factors for cancer have been historically verylt to identify, and those that are known are found inew patients and make up a small minority of cancer(4). Because the 3′UTR of a gene is a critical regula-egion, we have proposed that this region is likely tor variants, such as the KRAS-variant, that will beiated with a large proportion of cancer cases ande as powerful as gene-coding mutations in shapinge risk (14). Our findings support this hypothesis and

Figfromdem(maHBlessknoandlunfamKRof dwit

rce that the role ofr syndromes should

3. KRAS-variant–carrying families differRCA families. Cancer types and basicraphics in KRAS-variant–carryingd) versus BRCA1- and BRCA2-carryingfamilies. The KRAS-variant families areely to be Jewish (Ashkenazi status notthan BRCA1-mutant families (P = 0.003)re likely to have a family history ofncer as compared with BRCA2-mutant(P = 0.02); OC patients carrying the

variant are significantly older at the timenosis than BRCA1-mutant patients

such 3′UTR variants in familialbe intensively studied.

Cancer Research

Page 7

Disclo

F.J. Sdisclose

Ackn

We tthe YaleKennetKatsaro

Grant

Refe1. Jem

J C2. Cru

ries3. AC4. Str

tion5. An

cer6. Ph

Potion

7. Satioon

8. Qumocan

9. SoideGe

10. Hirgic

11. GoMe

12. Hadis

13. Esqcan

14. ChmeCa

15. Mi

MiRNA/KRAS–Based Genetic Marker of Ovarian Cancer Risk

www.a

sure of Potential Conflicts of Interest

lack and J.B. Weidhaas: ownership interest, MiraDx. The other authors NIH501/A3/ily CharthroughNIH gra

Theof pageaccorda

d no potential conflicts of interest.

owledgments

hank the Yale Department of Gynecological Oncology, the members of

Therapeutic Radiology Department, and Robert Burger. We also thank

and assistance and Dionyssios Rece

ntary site in the KRAS 3′UTR increases non-small cell cancer risk.ncer Res 2008;68:8535–40.shra P, Mishra P, Banerjee D, Bertino J. MiRSNPs or MiR-

pothe20

16. HumiCh

17. LumelowCa

18. DaanOn

19. Yahures

20. Jolet

21. Taofsho

22. Chsitesur

23. Risof15

24. NeKesm

25. IbaMoha

acrjournals.org

Support

grant R01 CA122728-01A2 and Italian Institute of Health (ISS) grants3 and 0027557 (A. Santin); NIH grant K08 CA124484, the Shannon Fam-itable Fund, and the RTOG Translational Research Program, fundedNational Cancer Institute grant U10CA21661 (J.B. Weidhaas); andnt CA131301-01A1 (F.J. Slack and J.B. Weidhaas).costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 02/26/2010; revised 05/14/2010; accepted 05/31/2010; published

h Kidd for his genetics expertise s and Sergio Pecorelli for use of samples. OnlineFirst 07/20/2010.

rencesal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancerlin 2008;58:71–96.m C. Intercepting pelvic cancer in the distal fallopian tube: theo-and realities. Mol Oncol 2009;3:165–70.

S. Cancer facts & figures 2006;2006.atton J, Gayther S, Russell P, et al. Contribution of BRCA1 muta-s to ovarian cancer. N Engl J Med 1997;226:1125–30.toniou A, Easton D. Risk prediction models for familial breast can-. Future Oncol 2006;2:257–74.aroah P, Antoniou A, Bobrow M, Zimmern R, Easton D, Ponder B.lygenic susceptibility to breast cancer and implications for preven-. Nat Genet 2002;31:33–6.saki R, Narisawa-Saito M, Yugawa T, et al. Oncogenic transforma-n of human ovarian surface epithelial cells with defined cellularcogenes. Carcinogenesis. Epub ahead of print 2009 Jan 6.aye L, Song H, Ramus S, et al. Tagging single-nucleotide poly-rphisms in candidate oncogenes and susceptibiity to ovariancer. Br J Cancer. Epub 2009.ng H, Ramus S, Tyrer J, et al. A genome-wide association studyntifies a new ovarian cancer susceptibility locus on 9p22.2. Natnet 2009. Advance online publication.schhorn J. Genomewide association studies—illuminating biolo-pathways. N Engl J Med 2009;360:1699–701.ldstein D. Common genetic variation and human traits. N Engl Jd 2009;360:1696–8.rdy J, Singleton A. Genomewide association studies and humanease. N Engl J Med 2009;360:1759–68.uela-Kerscher A, Slack FJ. Oncomirs: microRNAs with a role incer. Nat Rev Cancer 2006;6:259–69.in L, Ratner E, Leng S, et al. A SNP in a let-7 microRNA comple-

lymorphisms, new players in microRNA mediated regulation ofcell: introducing microRNA pharmacogenomics. Cell Cycle

08;7:853–8.Z, Liang J, Wang Z, et al. Common genetic variants in pre-

croRNAs were associated with increased risk of breast cancer ininese women. Hum Mutat 2009;30:79–84.L, Katsaros D, Rigault de la Longrais I, Sochirca O, Yu H. Hyper-thylation of let-7a-3 in epithelial ovarian cancer is associated withinsulin-like growth factor-II expression and favorable prognosis.

ncer Res 2007;67:10117–22.hiya N, Sherman-Baust C, Wang T-L, et al. MicroRNA expressiond identification of putative miRNA targets in ovarian cancer. PLOSe 2008;3:e2436.ng H, Kong W, He L, et al. MicroRNA expression profiling inman ovarian cancer: miR-214 incudes cell survival and cisplatinistance by targeting PTEN. Cancer Res 2008;68:425–33.hnson SM, Grosshans H, Shingara J, et al. RAS is regulated by the-7 microRNA family. Cell 2005;120:635–47.kamizawa J, Konishi H, Yanagisawa K, et al. Reduced expressionthe let-7 microRNAs in human lung cancers in association withrtened postoperative survival. Cancer Res 2004;64:3753–6.ristensen B, Moyer B, Avissar M, et al. A let-7 microRNA bindingpolymorphism in the KRAS 3′UTR is associatied with reduced

vival in oral cancers. Carcinogenesis. Epub ahead of print 2009.ch J, Bale A, Beck P, Zheng W. PGR +331 A/G and increased riskepithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev 2006;:1738–41.lson HH, Christensen BC, Plaza SL, Wiencke JK, Marsit CJ,lsey KT. KRAS mutation, KRAS-LCS6 polymorphism and non-all cell lung cancer. Lung Cancer. Epub ahead of print 2009.nez-Ventoso C, Yang M, Guo S, Robins H, Padgett R, Driscoll M.

dulated microRNA expression during adult life span in Caenor-bditis elegans. Aging Cell 2006;5:235–46.

Cancer Res; 70(16) August 15, 2010 6515